Human Immunodeficiency Virus (HIV), the causative agent of AIDS, continues to spread rapidly throughout the world. WHO and UNAIDS estimate that more than 60 million people have been infected with the virus since the beginning of the epidemic. At the end of 2004 more than 40 million individuals, mostly living in the developing countries, were infected with HIV. The inexorable spreading of the HIV pandemic and the resulting morbidity and mortality arising from AIDS in developing countries underscore the urgency for an effective, safe and inexpensive vaccine against AIDS.
Over the last 15-20 years, most of the efforts in HIV vaccine development have been focused on achieving sterilising immunity by targeting the Envelope protein (Env) of HIV responsible for the binding and entry of the virus, with the rationale of generating neutralising antibodies (NA) capable of protecting against infection (Wahren, 2002). However, results from pre-clinical and clinical trials, including the first phase III trial (AIDSVAX by VaxGen), in which no protection from primary infection in Caucasians has been observed, have been largely disappointing. This can be accounted for by the inability of such vaccines to elicit protective NA, owing to the high variability of Env (Myers, 1995), and hampering the recognition of relevant, mostly conformational, epitopes by the NA, and by the heavy glycosylation of gp120 that contributes to hiding critical (neutralising) env-epitopes (reviewed in Burton 1997).
More recently, other approaches have been developed aimed at inducing T-cell mediated responses against other HIV antigens. These approaches are aimed at controlling virus replication, which is achieved in the absence of sterilising immunity, providing protection from disease progression, and thus reducing virus transmission to healthy individuals. However, these approaches have also failed. An example of this is the recent trial by Merck, based on three HIV genes. This vaccine was created using a mixture of three components, each made with a replication-defective version of one of the common cold viruses, adenovirus type 5 (Ad5), which served as a carrier, or delivery vector, for the HIV gag, pol and nef genes. The vaccine did not prevent infection: in volunteers who received at least one dose of the three-dose vaccine series, 24 cases of HIV infection were observed in the 741 volunteers who received vaccine and 21 cases of HIV infection were observed in the 762 participants in the placebo group. In addition, the vaccine did not reduce the amount of virus in the bloodstream of those who became infected; HIV RNA levels approximately 8 to 12 weeks after diagnosis of infection were similar in the vaccine and the placebo arms.
A radically different approach is based on Tat, a key HIV regulatory gene and its protein product, Tat, as a vaccine candidate. Being a very early regulatory protein and playing a major role in HIV-1 replication and pathogenesis, Tat represents an optimal candidate for vaccine development (Ensoli, 1990, 1993 and 1994; Chang, 1997). Tat is a key viral regulatory protein produced very early after infection, even prior to HIV integration, and is necessary for viral gene expression (Arya, 1985; Fisher, 1986; Ensoli, 1993; Wu, 2001), as well as cell-to-cell virus transmission and disease progression. In fact, in the absence of Tat, no or negligible amounts of structural proteins are expressed and, therefore, no infectious virus is made. Further, Tat is released by the infected T lymphocytes in the extra-cellular milieu (Ensoli, 1990 and 1993; Chang, 1997) and enters both infected cells, where it promotes HIV-1 replication, and uninfected cells, where it causes activation or repression of cytokines and cellular genes controlling the cell cycle (Frankel and Pabo, 1988; Ensoli, 1993; Chang, 1995). This approach is aimed at inducing both antibodies neutralizing extracellular Tat and T cell responses against virus-infected cells.
Several studies suggest that an immune response to Tat has a protective role and may control the progression of the disease in vivo (Reiss, 1990; Rodman, 1993; Re, 1995; Zagury, 1998; Re, 2001). In particular, a higher prevalence of anti-Tat antibodies has been shown in asymptomatic HIV-infected individuals as compared to patients in advanced stages of the disease (Krone 1988, Demirhan 2000, Re 2001) and in non-progressors as compared to fast progressors (Zagury 1998). We recently performed a cross-sectional and longitudinal study in a cohort of 252 individuals with accurately estimated dates of seroconversion and a median follow-up of 7.2 years (Rezza, in press). The results of this study revealed a strong association between the presence of anti-Tat antibodies and a slower progression to the disease. In fact, the risk of developing AIDS or severe immunodeficiency was 60% lower for anti-Tat positive individuals than for anti-Tat negative individuals. The longitudinal analysis also indicated that individuals who are persistently anti-Tat positive have the lowest risk of disease progression, whereas those who are persistently anti-Tat negative have the highest risk of developing severe immunodeficiency. Notably, individuals who are transiently anti-Tat positive/negative have a nearly 70% lower risk as compared to those who are persistently negative, providing evidence that the presence of anti-Tat antibodies is predictive of slower disease progression (Rezza, in press).
The immunogenic regions of Tat are conserved (both B and T cell) across all epitope M subtypes (Myers, 1995). Our recent data, in fact, indicate an effective cross-recognition of Glade B strain-derived (BH-10) Tat protein from the HTLVIIIB lab-adapted virus strain (Butte, 2003), which was isolated about 20 years ago (Ratner, 1985) by individuals infected by viruses circulating in Africa and belonging to different HIV-1 clades, thereby reflecting the high degree of conservation of the corresponding Tat regions. Specifically, sera from Italian, Ugandan and South African patients who were mainly infected with A, B, C and D and to a lesser extent, F and G HIV-1 subtypes, recognised the BH-10 Tat protein at similar levels (i.e. prevalence and titres of anti-Tat antibodies). This observation is reinforced by the results of sequence conservation analysis, demonstrating that the predicted amino acid sequence of Tat is well conserved among the different circulating viruses belonging to distinct HIV-1 clades and presents a relatively high degree of homology with the BH-10 Tat sequence (Buttò, 2003).
Homology is specifically high in the first exon-encoded portion of Tat, and, particularly, in the 1-58 region, which contains the functional domains of Tat and most of the B, T-helper and CTL epitopes so far identified (Buttò, 2003). Furthermore, epitope mapping studies of the Tat-positive sera from Italian, Ugandan and South African patients using linear peptides from the same BH-10 Tat sequence indicate the same pattern of recognition and confirm that the amino terminal region contains the major B cell epitope of Tat, although a large portion of anti-Tat antibodies is represented by conformational antibodies, independently from the infecting virus strain (Buttò, 2003). These findings indicate that the overall identity of Tat is preserved also at the conformational level and provide strong formal evidence that a Tat-based vaccine may indeed represent a “universal” tool against HIV, since it is capable of inducing a broad immune response that is effective against different virus clades.
We have confirmed this hypothesis through preclinical studies performed in different animal models, including mice and cynomolgus monkeys, which demonstrated that vaccination with a biologically active Tat protein or tat DNA is safe, elicits a broad and specific immune response and, most importantly, induces a long-term protection in vaccinated monkeys against infection with a highly pathogenic virus (SHIV 89.6P), which causes AIDS and death in these monkeys (Cafaro, 1999, 2000 and 2001).
In particular, the native Tat protein induced Th-1 and CD8+ CTLs cellular immune responses and high titres of anti-Tat antibodies and blocked primary infection with the simian/human immunodeficiency virus (SHIV) 89.6P, as indicated by maintenance of the CD4+ T cell counts and lack of disease onset in cynomolgus monkeys (Cafaro, 1999, 2000 and 2001). Of note, protection was prolonged, since no signs of virus replication were found, either in peripheral blood mononuclear cells, or in lymph nodes of the protected animals, during the 2 years of follow up. Further, no residual virus hidden in resting cells was detected in any of the protected macaques either in the plasma or in lymph nodes, upon two boosts with tetanus toxoid, a stimulus known to activate virus replication. Long-term protection correlated with the presence of high and stable humoral and cellular (CD4 and CD8 T-cell responses) against Tat (Maggiorella 2004). Finally, a pilot study conducted in SHIV infected macaques indicated that vaccination with either Tat DNA or protein is safe also in monkeys with AIDS.
In addition to representing a valuable antigen for a HIV/AIDS vaccine, biologically active Tat also displays immunomodulatory features that make it an attractive adjuvant for other antigens. In fact, we have recently shown that monocyte-derived dendritic cells (MDDC), and to a much lesser extent macrophages, but not B lymphoblastoid cell lines or T cell blasts, efficiently and rapidly take up native but not oxidised-inactive Tat (Fanales-Belasio, 2002). Upon uptake, native Tat promotes MDDC maturation and activation (increased expression of MHC antigens and co-stimulatory molecules, production of Th-1 cytokines and chemokines), leading to a more efficient presentation of both allogeneic and exogenous soluble antigens (Fanales-Belasio, 2002). Very recent data indicate that these effects are all mediated by the induction of TNFα expression by native Tat (Fanales-Belasio, submitted).
Further, we have shown that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-1-binding CTL epitopes of heterologous antigens (Gavioli, 2004). Thus, the modifications of antigen processing and of the generation of CTL epitopes by Tat may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.
In conclusion, there is a growing body of evidence that indicates that biologically active Tat functions as both an antigen and a potent adjuvant since i) it induces MDDCs maturation and activation toward a Th1 inducing phenotype, ii) it gains access to the major histocompatibility complex (MHC) class I pathway of presentation (Moy et al., Mol Biotechnol, 1996; Kim et al., J Immunol 1997), and iii) it modulates the proteasome catalytic subunit composition, modifying the hierarchy of the CTL epitopes presented in favour of subdominant and cryptic epitopes. Taken together these features make Tat an optimal candidate for an HIV vaccine, alone or in combination with other antigens.
In seropositive patients, this should contribute to reducing HIV-1 replication and disease progression. In individuals exposed to the virus after vaccination the vaccine could modify the virus-host dynamics at the very beginning of the infection and this would impact on the depletion of critical immune cells and the evolution of the infection, since the accumulating evidence indicates that the level of viral load at the beginning of the infection is a strong indicator of progression to disease (Mellors, 1996; Watson, 1997; Lifson, 1997; Ten Haaft, 1998; Staprans, 1999).
This approach also has the advantage that HIV components, which would show up in current tests for HIV, are not used, and patients test HIV-negative.
It is essential that the biologically active form of Tat is used, since the preservation of the native conformation permits: the induction of an effective Th1 cellular immune response; the induction of antibodies directed against conformational epitopes; and is necessary for the retention of Tat's adjuvant properties.
The Tat protein of HIV-1 (HTLV-IIIB strain, clone BH-10) is a molecule of 86 amino acids encoded by two exons. The product of the first exon is sufficient for the transactivation of the HIV-1 promoter. This region contains four domains including the amino terminal domain (aa 1-21), the cystein-rich domain (aa 22-37), the core (aa 38-48) and the basic domain (49-57). The cystein-rich region is necessary for zinc ion-mediated dimer formation and represents the transactivation domain. The basic region, rich in lysine and arginine residues, is required for nuclear localisation and can bind specifically to its RNA target, the transactivation response element (TAR) in the LTR (Hauber, 1989; Ruben, 1989; Roy, 1990; Chang, 1995). The C-terminal 14 amino acids of Tat that are encoded by exon 2 is not necessary for HIV-1 LTR transactivation but contains the arginine-glycine-aspartate (RGD) sequence which is a motif present in extracellular matrix proteins (Barillari, 1993; Brake, 1990). This region and the basic domain are required for the interaction of extracellular Tat with heparan sulphate proteoglycans and with cell surface molecules of the integrin family, respectively, and mediate uptake of Tat by dendritic cells (DC) (Fanales-Belasio, 2002).
In fact, biologically active Tat is selectively and efficiently taken up and processed by monocyte-derived DC, inducing their maturation and promoting their capacity to present, antigens, eliciting immune responses toward a Th1 pattern, and increasing T cell responses to other antigens. These functions are exerted only by the biologically active Tat protein and are abolished or greatly hampered after oxidation/inactivation of the protein (Fanales-Belasio, 2002).
Attempted production of Tat by conventional culture of recombinant hosts has been found to yield only biologically inactive Tat and is, thus, useless for the production of Tat on a commercial scale, as recovery of the biological properties is possible only after total denaturation and proper refolding of the product. Denaturation and refolding methods require the use of solvents not permitted in biologics intended for human use, so that these methods are neither useful nor can they serve as a guide for the production of biologically active Tat.